Cloning, purification and characterization of a thermostable β- galactosidase from Bacillus licheniformis strain KG9
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Tarih
2015
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Cellular and Molecular Biology Association
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
A thermo- and alkalitolerant Bacillus licheniformis KG9 isolated from Taslidere hot water spring in Batman/Turkey was found to produce a thermostable ?-galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative ?-galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding ?-galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the ?-galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant ?-galactosidase was produced in Escherichia coli. Of the four ?-galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, ?-galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 °C and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho-nitrophenyl-?-D-galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 ?mol/min and 1.55 ?mol/min with oNPG and lactose, respectively.
Açıklama
Anahtar Kelimeler
Bacillus licheniformis, Enzyme activity, Purification, Recombinant Dna, Thermophiles, ?-Galactosidase
Kaynak
Cellular and Molecular Biology
WoS Q Değeri
Scopus Q Değeri
Q4
Cilt
61
Sayı
3
Künye
Bekler, F. M., Stougaard, P., Güven, K., Güven, R. G. ve Acer, Ö. (2015). Cloning, purification and characterization of a thermostable β- galactosidase from Bacillus licheniformis strain KG9. Cellular and Molecular Biology, 61(3), 71-78.
